The present invention relates to assays for the determination of analytes in fluids.
Many types of assay elements for the rapid analysis of analytes present in biological fluids are known in the art. Of particular interest are dry multilayer analytical elements to which the sample, e.g., a drop of blood, serum or plasma, is applied and allowed to migrate or diffuse to a reagent layer or layers. As a result of the interaction between the analyte and the reagent(s) present, a detectable change is brought about in the element corresponding to the presence of the analyte in the sample. The detectable change can be a color change which may be evaluated visually or read spectrophotometrically such as with a densitometer. In another scheme based on the presence of fluorescent-labeled biologically active species, a fluorescent output signal can be generated and read spectrofluorometrically. Such assay elements are of great interest because they can be adapted for use in automated analytical instruments.
In the automated analytical instruments a sample of a test fluid is typically provided in a sample cup and all of the assay method steps including pipetting of a measured volume of the sample onto an assay element, incubation and readout of the signal obtained as a result of the interactions(s) between the reagent(s) and the sample analyte are carried out automatically. The assay element is typically transported from one station, e.g. the pipetting station, to another, e.g. the optical read station, by a transport means such as a rotating carousel to enable the test steps to be carried out automatically.
Such automated analytical instruments are capable of processing many assay elements rapidly and it is necessary to achieve a very high level of precision for these assays. However, imprecisions in the results obtained can be caused by a number of factors. For example, any element to element variation in the distance from the optical readout apparatus to the signal-generating species when readout of the signal is carried out will introduce imprecision into the results as will any element to element variation in the thickness of the layer in which the signal-generating species resides when it is read.
The reagent layer(s) in thin film multilayer assay elements may be extremely thin, that is, on the order of about 0.01 mm or less. Accordingly, although such layers can be coated with a very high degree of precision nevertheless some slight variation in the thickness of the reagent layers will exist on an element to element basis. Similarly, although the transport means e.g. a carousel, for the assay elements can be engineered within very exact tolerances, nevertheless there will exist some slight variations in the instrument position response for the respective assay element positions on the transport means.
It is desirable therefore to have a method for compensating for signal imprecisions caused by variations in reagent levels from element to element and variations in instrument position response as well those caused by other factors.